ML-236B Derivatives

ABSTRACT

Compounds of formula (I): ##STR1## (wherein R represents a group of formula ##STR2## and the corresponding ring-closed lactones, salts (especially alkali metal salts) and esters (especially C 1  -C 5  alkyl esters) thereof may be prepared by subjecting ML-236B, or ML-236B carboxylic acid or a salt or ester thereof to enzymatic hydroxylation, which may be effected by means of microorganisms of the genera Mucor, Rhizopus, Zygorynchus, Circinella, Actinomucor, Gongronella, Phycomyces, Martierella, Pycnoporus, Rhizoctonia, Absidia, Cunninghamella, Syncephalastrum and Streptomyces, or cell-free, enzyme-containing extracts from said microorganisms. The compounds are capable of inhibiting biosynthesis of cholesterol and are thus useful in the treatment of hypercholesteraemia.

This is a division of application Ser. No. 270,846 filed June 5, 1981now U.S. Pat. No. 4,346,227 issued Aug. 24, 1982.

BACKGROUND TO THE INVENTION

The present invention relates to a series of new derivatives of theknown compound ML-236B, to processes for their preparation and topharmaceutical compositions containing them.

ML-236B, which has the following chemical structure: ##STR3## isdisclosed in U.S. Patent Specification No. 3,983,140. It has beenisolated and purified from the metabolic products of microorganisms ofthe genus Penicillium, especially Penicillium citrinum, a species ofblue mould. It has been shown to inhibit the biosynthesis of cholesterolby enzymes or cultured cells separated from experimental animals bycompeting with the rate-limiting enzyme active in the biosynthesis ofcholesterol, namely 3-hydroxy-3-methylglutaryl-coenzyme A reductase and,as a result, significantly reduces serum cholesterol levels of animals[Journal of Antibiotics, 29, 1346 (1976)]. A number of compoundsstructurally related to ML-236B have also been discovered and found topossess the ability to inhibit the biosynthesis of cholesterol.

We have now discovered a series of new compounds, which may be preparedby the enzymatic hydroxylation of ML-236B or of derivatives thereof, andwhich possess an ability to inhibit the biosynthesis of cholesterolwhich is at least comparable with, and in some instances substantiallyexceeds, that of ML-236B itself.

BRIEF SUMMARY OF INVENTION

The compounds of the present invention are those hydroxycarboxylic acidsof formula (I): ##STR4## (in which R represents a group of formula##STR5## and ring-closed lactones, salts and esters thereof.

The invention also provides a process for preparing a compound offormula (I), or a ring-closed lactone, salt or ester thereof by theenzymatic hydroxylation of ML-236B, or ML-236B carboxylic acid, or asalt or ester thereof.

ML-236B carboxylic acid has the formula ##STR6##

DETAILED DESCRIPTION OF INVENTION

One class of compounds of the present invention are those compounds offormula (II): ##STR7## (in which R¹ represents a hydrogen atom or a C₁-C₅ alkyl group), pharmaceutically acceptable salts of the acid whereinR¹ represents a hydrogen atom, and the corresponding lactone of formula(III): ##STR8## In view of the number of asymmetric carbon atoms inthese compounds, a variety of geometric isomers are possible. Of these,the most important isomers are as follows:

Compounds of formula (IV): ##STR9## (in which R¹ is as defined above)and pharmaceutically acceptable salts of the acid wherein R¹ representsa hydrogen atom, and the corresponding lactone of formula (V): ##STR10##and compounds of formula (VI): ##STR11## (in which R¹ is as definedabove), and pharmaceutically acceptable salts of the acid wherein R¹represents a hydrogen atom and the corresponding lactone of formula(VII): ##STR12##

The hydroxy-carboxylic acid of formula (IV) in which R¹ represents ahydrogen atom is herein referred to as M-4 and derivatives of this acid,specifically the salts and esters, are named as derivatives of M-4,whilst the corresponding lactone of formula (V) is herein referred to asM-4 lactone. Similarly, the hydroxy-carboxylic acid of formula (VI) inwhich R¹ represents a hydrogen atom is referred to as M-4' andderivatives of this acid are referred to as derivatives of M-4', whilstthe corresponding lactone of formula (VII) is referred to as M-4'lactone.

Another preferred class of compounds of the invention are thosecompounds of formula (VIII): ##STR13## (in which R¹ is as definedabove), and pharmaceutically acceptable salts of the acid in which R¹represents a hydrogen atom, and the corresponding lactone of formula(IX): ##STR14##

A variety of geometric isomers of these compounds are also possible, themost important being the following:

Compounds of formula (X): ##STR15## (in which R¹ is as defined above),and pharmaceutically acceptable salts of the acid in which R¹ representsa hydrogen atom and the corresponding lactone of formula (XI): ##STR16##and compounds of formula (XII): ##STR17## (in which R¹ is as definedabove), and pharmaceutically acceptable salts of the acid in which R¹represents a hydrogen atom and the corresponding lactone of formula(XIII): ##STR18##

The acid of formula (X) is herein referred to as IsoM-4 and itsderivatives, such as salts and esters, are named as derivatives ofIsoM-4, whilst the corresponding lactone of formula (XI) is hereinreferred to as IsoM-4 lactone. The acid of formula (XII) in which R¹represents a hydrogen atom is herein referred to IsoM-4', and itsderivatives are named as derivatives of IsoM-4', whilst itscorresponding lactone of formula (XIII) is herein referred to as IsoM-4'lactone.

Of the esters of the hydroxy-carboxylic acids of formula (I), the C₁ -C₅alkyl esters are preferred. These alkyl groups may be straight orbranched-chain groups and include, for example the methyl, ethyl,propyl, isopropyl, butyl and isobutyl groups, of which the methyl groupis particularly preferred.

The hydroxy-carboxylic acids will also form salts with a variety ofcations, particularly metals and most preferably alkali metals, such assodium or potassium. The sodium salts are most preferred.

Of the compounds of the invention, the most preferred compounds are M-4lactone, M-4 sodium salt, M-4 methyl ester, IsoM-4' lactone, IsoM-4'sodium salt and IsoM-4' methyl ester, M-4 sodium salt being particularlypreferred.

The compounds of the invention may be prepared by the enzymatichydroxylation of ML-236B or of a derivative thereof, specificallyML-236B carboxylic acid or a salt or ester thereof.

This enzymatic hydroxylation may be effected as part of the mammalianmetabolism of ML-236B or a derivative thereof, for example byadministering ML-236B to a suitable animal, collecting a metabolicproduct, eg. urine, and then separating the desired compound orcompounds of the invention from the urine. Alternatively, the liver oran enzyme-containing extract from the liver may be used instead of theliving animal. However, processes employing the animal metabolism oranimal products have a relatively low productivity and are difficult toeffect reproducibly. Accordingly, we prefer to employ microorganisms orenzyme-containing extracts from the microorganisms.

Accordingly, the process of the present invention is preferably effectedusing a microorganism capable of converting ML-236B or a derivativethereof to a compound of the present invention or using anenzyme-containing extract of such a microorganism. Particularlypreferred microorganisms are those of the following genera: Mucor,Rhizopus, Zygorynchus, Circinella, Actinomucor, Gongronella, Phycomyces,Martierella, Pycnoporus, Rhizoctonia, Absidia, Cunninghamella,Syncephalasporum and Streptomyces. In particular the following speciesare preferred:

Absidia coerulea

Cunninghamella echinulata

Syncephal strum racemosum

Streptomyces roseochromogenus

Mucor hiemalis f. hiemalis

Mucor bacilliformis

Mucor circinelloides f. circinelloides

Mucor hiemalis f. corticolus

Mucor dimorphosporus

Mucor fragilis

Mucor genevensis

Mucor globosus

Mucor circinelloides f. griseo-cyanus

Mucor heterosporus

Mucor spinescens

Rhizopus chinensis

Rhizopus circinans

Rhizopus arrhizus

Zygorynchus moelleri

Circinella muscae

Circinella rigida

Circinella umbellata

Actinomucor elegans

Phycomyces blakesleeanus

Martierella isabellina

Gongronella butleri

Pycnoporus coccineus

Rhizoctonia solani

Syncephalastrum nigricans

Absidia glauca var. paradoxa

Amongst strains of the above species, the following are particularlypreferred:

Absidia coerulea IFO-4423

Cunninghamella echinulata IFO-4445

Cunninghamella echinulata IFO-4444

Cunninghamella echinulata ATCC-9244

Syncephalastrum racemosum IFO-4814

Syncephalastrum racemosum IFO-4828

Streptomyces roseochromogenus NRRL-1233

Streptomyces roseochromogenus IFO-3363

Streptomyces roseochromogenus IFO-3411

Mucor hiemalis f. hiemalis IFO-5834

Mucor hiemalis f. hiemalis IFO-5303

Mucor hiemalis f. hiemalis IFO-8567

Mucor hiemalis f. hiemalis IFO-8449

Mucor hiemalis f. hiemalis IFO-8448

Mucor hiemalis f. hiemalis IFO-8565

Mucor hiemalis f. hiemalis CBS-117.08

Mucor hiemalis f. hiemalis CBS-109.19

Mucor hiemalis f. hiemalis CBS-200.28

Mucor hiemalis f. hiemalis CBS-242.35

Mucor hiemalis f. hiemalis CBS-110.19

Mucor hiemalis f. hiemalis CBS-201.65

Mucor bacilliformis NRRL-2346

Mucor circinelloides f. circinelloides IFO-4554

Mucor circinelloides f. circinelloides IFO-5775

Mucor hiemalis f. corticolus NRRL-12473

Mucor dimorphosporus IFO-4556

Mucor fragilis CBS-236.35

Mucor genevensis IFO-4585

Mucor globosus NRRL 12474

Mucor circinelloides f. griseo-cyanus IFO-4563

Mucor heterosporus NRRL-3154

Mucor spinescens IAM-6071

Rhizopus chinensis IFO-4772

Rhizopus circinans ATCC-1225

Rhizopus arrhizus ATCC-11145

Zygorynchus moelleri IFO-4833

Circinella muscae IFO-4457

Circinella rigida NRRL-2341

Circinella umbellata NRRL-1713

Circinella umbellata IFO-4452

Circinella umbellata IFO-5842

Phycomyces blakesleeanus NRRL-12475

Martierella isabellina IFO-6739

Gongronella butleri IFO-8080

Pycnoporus coccineus NRRL-12476

Rhizoctonia solani NRRL-12477

Syncephalastrum nigricans NRRL-12478

Syncephalastrum nigricans NRRL-12479

Syncephalastrum nigricans NRRL-12480

Absidia glauca var. paradoxa IFO-4431

Actinomucor elegans ATCC-6476

The microorganisms listed above are available from International CultureCollections, as indicated by the codes appended to their accessionnumbers, which codes have the following meanings.

IFO=Institute for Fermentation, Osaka, Japan

NRRL=Novthern Utilization Research and Development Division, U.S.Department of Agriculture, Peoria, Ill., USA

CBS=Centraal bureau voor Schimmelcultures, Netherlands

IAM=Institute of Applied Microbiology, Tokyo, Japan

ATCC=American Type Culture Collection, Maryland, USA.

Of the species noted above, the following are particularly preferred:

Absidia coerulea

Cunninghamella echinulata

Syncephalastrum racemosum

Mucor hiemalis f. hiemalis

Mucor bacilliformis

Mucor circinalloides f. circinelloides

Mucor hiemalis f. corticolus

Mucor dimorphosporus

Mucor fragilis

Mucor genevensis

Mucor globosus

Mucor circinelloides f. griseo-cyanus

Mucor heterosporus

Mucor spinescens

Pycnoporus coccineus

Rhizoctonia solani

Syncephalastrum nigricans

and the following are particularly preferred strains of the species:

Absidia coerulea IFO-4423

Cunninghamella echinulata IFO-4445

Cunninghamella echinulata IFO-4444

Cunninghamella echinulata ATCC-9244

Syncephalastrum racemosum IFO-4814

Syncephalastrum racemosum IFO-4828

Mucor hiemalis f. hiemalis IFO-5834

Mucor hiemalis f. hiemalis IFO-5303

Mucor hiemalis f. hiemalis IFO-8567

Mucor hiemalis f. hiemalis IFO-8449

Mucor hiemalis f. hiemalis IFO-8448

Mucor hiemalis f. hiemalis IFO-8565

Mucor hiemalis f. hiemalis CBS-117.08

Mucor hiemalis f. hiemalis CBS-109.19

Mucor hiemalis f. hiemalis CBS-200.28

Mucor hiemalis f. hiemalis CBS-242.35

Mucor hiemalis f. hiemalis CBS-110.19

Mucor hiemalis f. hiemalis CBS-201.65

Mucor bacilliformis NRRL-2346

Mucor circinelloides f. circinelloides IFO-4554

Mucor circinelloides f. circinelloides IFO-5775

Mucor hiemalis f. corticolus NRRL-12473

Mucor dimorphosporus IFO-4556

Mucor fragilis CBS-236.35

Mucor genevensis IFO-4585

Mucor globosus NRRL 12474

Mucor circinelloides f. griseo-cyanus IFO-4563

Mucor heterosporus NRRL-3154

Mucor spinescens IAM-6071

Pycnoporus coccineus NRRL-12476

Rhizoctonia solani NRRL-12477

Syncephalastrum nigricans NRRL-12478

Syncephalastrum nigricans NRRL-12479

Syncephalastrum nigricans NRRL-12480

For the preparation of compounds of formulae (IV) and (V) and theirsalts, the following species are preferred:

Mucor hiemalis f. hiemali

Mucor circinelloides f. circinelloides

Mucor fragilis

Mucor genevensis

Mucor circinelloides f. griseo-cyanus

Pycnoporus coccineus

Rhizoctonia solani.

For the preparation of compounds of formula (VI) and (VIII) and theirsalts, the species Syncephalastrum nigricans and Syncephalastrumracemosum are preferred.

For the preparation of compounds of formula (VIII) and (IX) and theirsalts, the species Absidia coerulea and Cunninghamella echinulata arepreferred.

Of all of the species listed above, Mucor hiemalis f. hiemalis isparticularly preferred since it is able to convert ML-236B and itsderivatives to the desired compounds of formula (I) at a conversion of90% or even higher.

Conversion of ML-236B or derivatives thereof of compounds of formula (I)may be achieved by contacting the complete cellular microorganism or, insome cases, a cell-free extract from the microorganism with ML-236B or aderivative thereof. The form of the compound produced will depend uponthe culture conditions and the form of microorganism employed. This, forexample, if the complete cellular microorganism is cultivated in thepresence of ML-236B or a derivative thereof, the product will be thecarboxylic acid, the lactone or alkali metal salt, depending upon theculture conditions, particularly the pH. On the other hand, if theML-236B or derivative thereof is simply contacted with a restingcellular system or with a cell-free extract, the compound of theinvention is obtained in the form of an alkali metal salt.

The progress of the conversion reaction may be determined by assayingsamples of the reaction mixture during the course of the reaction todetermine the degree of conversion. For example, the presence of M-4lactone may be assayed by liquid chromatography employing as a carrier μBondapak C₁₈ (manufactured by Waters Co. U.S.A.) and as the solvent 62%v/v aqueous methanol at the rate of 1 ml/minute. When detected using itsultraviolet absorption at 237 nm, M-4 gives a peak at a retention timeof 10 minutes, and this may be used for the assay. Similar techniquesare available for assaying the other compounds of the invention.

Where the microorganisms are to be cultivated in the presence of ML-236Bor a derivative thereof to product the compounds of the invention, theculture conditions and culture media employed will be chosen havingregard to the particular microorganism to be cultivated. Since thespecies of microorganism proposed for use in the process of the presentinvention are well known, culture conditions and culture media for usewith these microorganisms are also well known.

The compounds of the invention may be separated from the reactionmixture by conventional means, for example by filtering off microbialcells (if necessary) and then subjecting the remaining mixture to anycombination of thin layer chromatography, column chromatography or highperformance liquid chromatography. The various compounds of theinvention, where two or more are prepared together, may be separatedfrom each other in the course of one or more of these chromatographicpurification steps.

In addition to the compounds of the invention, there may, in some cases,also be prepared a compound which we have designated M-3 and which isknown under the name 3',5'-dihydroxy(dihydro-ML-236B) in a copendingapplication Ser. No. 256,243, filed Apr. 21, 1981 now U.S. Pat. No.4,361,515 entitled "Hydronaphthalene Derivatives, their Preparation andUse". This may also be separated in the same way.

We have found that the compounds of the invention give a 50% inhibitionof cholesterol biosynthesis at concentrations comparable with, or, insome cases, significantly less than, the concentrations required byML-236B and certain other similar known compounds. The inhibitoryactivities of the compounds of the invention, in terms of theconcentration in μg/ml required to inhibit cholesterol biosynthesis by50% [measured by the method described in the Journal of BiologicalChemistry, 234, 2835 (1959)] are as follows:

    ______________________________________                                        M-4 methyl ester       0.001                                                  M-4 sodium salt        0.0008                                                 M-4 lactone            0.016                                                  IsoM-4' methyl ester   0.007                                                  IsoM-4' lactone        0.013                                                  M-4' lactone           0.019                                                  M-4' sodium salt       0.00049                                                ML-236B                0.01                                                   ______________________________________                                    

The invention is further illustrated by the following Examples.

EXAMPLE 1 Preparation of M-4 lactone

Twenty 500 ml Sakaguchi flasks, each containing 100 ml of a mediumhaving the composition described below, were inoculated with spores ofAbsidia coerulea IFO 4423. The flasks were subjected to shaking cultureat 26° C. and 120 Strokes/min (s.p.m.) for 2 days. At the end of thistime, the sodium salt of ML-236B was added to each of the flasks to afinal concentration of 0.05% w/v. Cultivation was continued at 26° C.and 120 s.p.m. for a further 5 days.

The composition of the medium was (percentages are w/v):

    ______________________________________                                        Glucose            2.0%                                                       K.sub.2 HPO.sub.4  0.15%                                                      MgSO.sub.4.7H.sub.2 O                                                                            0.15%                                                      NH.sub.4 NO.sub.3  0.1%                                                       Peptone            0.1%                                                       Corn steep liquor  0.2%                                                       Yeast extract      0.1%                                                       ZnSO.sub.4.7H.sub.2 O                                                                            0.001%                                                     Tap water          the balance                                                                   (adjusted to pH 7.0).                                      ______________________________________                                    

After completion of the cultivation, the reaction liquor was filtered,and the filtrate was adjusted with trifluoroacetic acid to pH 3. Theresulting mixture was extracted with three 1 liter portions of ethylacetate, to give extracts containing M-4. This compound shows an Rfvalue of 0.45 on thin layer chromatography (TLC) (Plate: Merck silicagel Art 5715; solvent: a 50:50:3 by volume mixture of benzene, acetoneand acetic acid). The combined extracts were washed with saturatedaqueous sodium chloride, and then a catalytic amount of trifluoroaceticacid was added for lactonization. The resulting mixture was then washedwith a 1% w/v aqueous solution of sodium bicarbonate, dried overanhydrous sodium sulphate and evaporated under reduced pressure todryness. The residue was subjected to preparative liquid chromatography,System 500 using a Prep PAK-500/C₁₈ cartridge manufactured by WatersAssociates (Prep PAK is a Trade Mark). Purification with a 55% v/vaqueous methanol system yielded 50.1 mg of M-4 lactone.

M-4 lactone has the following physical properties.

(1) Nuclear Magnetic Resonance Spectrum:

The NMR spectrum measured at 60 MHz in deuterochloroform usingtetramethylsilane as the internal standard is shown in FIG. 1 of theaccompanying drawings.

(2) Ultraviolet absorption spectrum (methanol solution) λ_(max) nm:

230; 236.7; 244.6.

(3) Infrared absorption spectrum (liquid film) ν cm⁻¹ :

3400, 2950, 1725.

(4) Thin layer chromatography:

TLC plate: Merck silica gel Art 5715;

Solvent: benzene, acetone, acetic acid (50:50:3 by volume);

Rf value: 0.62.

EXAMPLE 2

48 mg of M-4 lactone were prepared following the same procedures as inExample 1, but using Cunninghamella echinulata IFO 4445.

EXAMPLE 3

30 mg of M-4 lactone were prepared following the same procedures as inExample 1, but using Streptomyces roseochromogenus NRRL 1233.

EXAMPLE 4

5 mg of M-4 lactone were prepared following the same procedures as inExample 1, but using Syncephalastrum racemosum IFO 4814.

EXAMPLE 5

6 mg of M-4 lactone were prepared following the same procedures as inExample 1, but using Syncephalastrum racemosum IFO 4828.

EXAMPLE 6 Preparation of IsoM-4'methyl ester

Twenty 500 ml Sakaguchi flasks, each containing 100 ml of a mediumhaving the composition described below, were inoculated with spores ofAbsidia coerulea IFO 4423. The flasks were subjected to shaking cultureat 120 s.p.m. and 26° C. for 2 days. At the end of this time, the sodiumsalt of ML-236B was added to each of the flasks to a final concentrationof 0.05% w/v. Cultivation was continued at 120 s.p.m. and 26° C. for afurther 5 days.

The composition of the medium was (percentages are w/v):

    ______________________________________                                        Glucose            2.0%                                                       K.sub.2 HPO.sub.4  0.15%                                                      MgSO.sub.4.7H.sub.2 O                                                                            0.15%                                                      NH.sub.4 NO.sub.3  0.1%                                                       Peptone            0.1%                                                       Corn steep liquor  0.2%                                                       Yeast extract      0.1%                                                       ZnSO.sub.4.7H.sub.2 O                                                                            0.001%                                                     Tap water          the balance                                                                   (adjusted to pH 7.0).                                      ______________________________________                                    

After completion of the cultivation, the reaction liquor was filtered,and the filtrate was adjusted with trifluoroacetic acid to pH 3. Theresulting mixture was extracted with three 1 liter portions of ethylacetate to give extracts containing IsoM-4'. This compound has an Rfvalue of 0.45 on thin layer chromatography (plate: Merck silica gel Art5715; solvent: a 50:50:3 by volume mixture of benzene, acetone andacetic acid). The extract was washed with a saturated aqueous solutionof sodium chloride, and then an ethereal solution of diazomethane wasadded. The mixture was allowed to stand for 30 minutes and thenevaporated under reduced pressure to dryness. The residue was placed ona Lobar column (Merck Si 60, Size A) and purified using as the solventsystem a 1:1 by volume mixture of benzene and ethyl acetate. There wereobtained 200 mg of an IsoM-4'methyl ester fraction. This fraction wasfurther purified on a Lobar column (Merck RP-8, Size A) using 35% v/vaqueous acetonitrile as the eluent to give 78 mg of pure IsoM-4'methylester, having the following characteristics:

(1) Nuclear Magnetic Resonance Spectrum:

The NMR spectrum measured at 100 MHz in deuterochloroform usingtetramethylsilane as the internal standard is shown in FIG. 2 of theaccompanying drawings.

(2) Mass spectrum:

Measurement was made [after silylation withN,O-bis(trimethylsilyl)trifluoroacetamide] using a mass spectrometer,type D-300 manufactured by Nippon Electronics.

M/e: 654 (M⁺), 552, 462, 372, 272, 233, 231.

(3) Ultraviolet absorption spectrum (methanol solution) λ_(max) nm:

229; 234.8; 244.5.

(4) Infrared absorption spectrum (liquid film):

As shown in FIG. 3 of the accompanying drawings.

(5) Thin layer chromatography:

TLC plate: Merck silica gel Art 5715;

Solvent: benzene, acetone (1:1 by volume);

Rf value: 0.88.

By operating as described above but replacing the diazomethane byanother appropriate diazoalkane, it is possible to produce other estersof IsoM-4'.

EXAMPLE 7 Preparation of IsoM-4'lactone

The procedure described in Example 6 was repeated up to and includingextraction with ethyl acetate to give extracts containing IsoM-4'. Thecombined extracts were washed with a saturated aqueous solution ofsodium chloride and then evaporated to dryness to give the lactoneproduct. The resulting residue was placed on a Lobar column (Merck Si60, Size A) and purified using as the solvent system a 1:1 by volumemixture of benzene and ethyl acetate, to afford 198 mg of IsoM-4'lactone. This product was further purified by means of a Lobar column(Merck RP-8, Size A) eluted with 35% v/v aqueous acetonitrile, to give82 mg of pure IsoM-4 lactone, having the following characteristics:

(1) Nuclear Magnetic Resonance Spectrum:

The NMR spectrum measured at 100 MHz in deuterochloroform usingtetramethylsilane as the internal standard is shown in FIG. 4 of theaccompanying drawings.

(2) Ultraviolet absorption spectrum (methanol solution) λ_(max) nm:

229; 234.8; 244.5.

(3) Infrared absorption spectrum (liquid film):

As shown in FIG. 5 of the accompanying drawings.

EXAMPLE 8

63 mg of IsoM-4'lactone were prepared, following the same procedures asin Example 7, but using Cunninghamella echinulata IFO 4445.

EXAMPLE 9

24 mg of IsoM-4'lactone were prepared, following the same procedures asin Example 7, but using Syncephalastrum racemosum IFO 4814.

EXAMPLE 10

35 mg of IsoM-4'lactone were prepared, following the same procedures asin Example 7, but using Syncephalastrum racemonsum IFO 4828.

EXAMPLE 11

12 mg of IsoM-4'lactone were produced according to the process describedin Example 7, but using Streptomyces roseochromogenus NRRL 1233.

EXAMPLE 12 Preparation of IsoM-4'sodium salt

In a small amount of acetone were dissolved 10 mg of IsoM-4' lactone. Tothe solution was added an equivalent amount of sodium hydroxide and themixture was allowed to stand for 1 hour. The pH of the resulting mixturewas adjusted with 0.1 N hydrochloric acid to a value of 8.0. The acetonewas then distilled off, and the residue was placed on an XAD-20 column(about 20 ml). The column was washed with distilled water and theneluted with 50 ml of 50% v/v aqueous acetone. The acetone was againdistilled off, and the residue was freeze-dried to afford 6 mg ofIsoM-4'sodium salt, having the following characteristics:

(1) Ultraviolet absorption spectrum (methanol solution) λ_(max) nm:

229 (shoulder); 235; 245 (shoulder).

(2) Infrared absorption spectrum (KBr) νcm⁻¹ :

3400, 2850, 1710, 1580.

(3) Thin layer chromatography:

TLC plate: Merck silica gel Art 5715;

Solvent: benzene, acetone, acetic acid (50:50:3 by volume);

Rf value: 0.45.

EXAMPLE 13 Preparation of M-4 methyl ester

Twenty 500 ml Sakaguchi flasks, each containing 100 ml of a medium ofthe same composition as shown in Example 1, were inoculated with sporesof Absidia coerulea IFO 4423. The flasks were subjected to shakingculture at 26° C. and 120 s.p.m. for 2 days. The sodium salt of ML-236Bwas then added to each of the flasks to a final concentration of 0.05%w/v. Cultivation was continued at 26° C. and 120 s.p.m. for a further 5days.

After completion of the cultivation, the reaction liquor was filtered,and the filtrate was adjusted with trifluoroacetic acid to pH 3. Theresulting mixture was extracted with three 1 liter portions of ethylacetate, to give extracts containing M-3, M-4 and IsoM-4'. Both M-4 andIsoM-4' show an Rf value of 0.45 on thin layer chromatography (Plate:Merck silica gel Art 5715; solvent: a 50:50:3 by volume mixture ofbenzene, acetone and acetic acid). The combined extracts were washedwith saturated aqueous sodium chloride, and then an ethereal solution ofdiazomethane was added. The mixture was allowed to stand for 30 minutesand then evaporated under reduced pressure to dryness. When the residuewas placed on a Lobar column (Merck Si 60, Size A), and purification waseffected using a 1:1 by volume mixture of benzene and ethyl acetate, afraction containing IsoM-4'methyl ester and a fraction containing M-4methyl ester were separated. There was obtained 185.3 mg of the latteractive fraction, from which 20 mg of pure M-4 methyl ester were obtainedas a colourless oil by using a Lobar column (Merck RP-8, Size A) andeluting with 35% v/v aqueous acetonitrile.

M-4 methyl ester has the following characteristics:

(1) Nuclear Magnetic Resonance Spectrum:

Measurement was made at 200 MHz in deuterochloroform usingtetramethylsilane as the internal standard.

δ ppm:

0.88 (3H, triplet, J=7.3 Hz);

0.89 (3H, doublet, J=6.5 Hz);

1.12 (3H, doublet, J=6.8 Hz);

1.1-1.7 (10H, multiplet);

2.34 (1H, sextuplet, J=7 Hz);

2.3-2.5 (2H, multiplet);

2.49 (2H, doublet, J=6.4 Hz);

2.58 (1H, multiplet);

3.72 (3H, singlet);

3.78 (1H, multiplet);

4.25 (1H, quintet, J=7 Hz);

4.4 (1H, multiplet);

5.42 (1H, multiplet);

5.56 (1H, multiplet);

5.90 (1H, doubled doublet, J=9.8 and 5.6 Hz);

5.99 (1H, doublet, J=9.8 Hz).

(2) Mass spectrum:

Measurement was made [after silylation withN,O-bis(trimethylsilyl)trifluoroacetamide] using a mass spectrometer,type D-300 manufactured by Nippon Electronics.

M/e: 654 (M⁺), 552, 462, 372, 290, 272, 233, 231.

(3) Ultraviolet absorption spectrum (ethanol solution) λ_(max) nm:

230.1; 237.3; 246.4.

(4) Infrared absorption spectrum (liquid film) ν cm⁻¹ :

3400, 2950, 1730.

(5) Thin layer chromatography:

TLC plate: Merck silica gel Art 5715;

Solvent: benzene and acetone (1:1 by volume);

Rf value: 0.88.

By operating as described above but replacing the diazomethane byanother appropriate diazoalkane, it is possible to produce other estersof M-4.

EXAMPLE 14 Preparation of Sodium Salts of M-4 and IsoM-4'

The procedure described in Example 1 except that Na'₂ HPO₄ was usedinstead of the K₂ HPO₄ was repeated up to and including filtration ofthe reaction liquor. The filtrate was then adsorbed on an HP-20 column(manufactured by Mitsubishi Chemical Industries). After washing thecolumn with water, fractions containing M-4 sodium salt, IsoM-4'sodiumsalt and M-3 sodium salt were eluted with 50% v/v aqueous acetone. Theactive fractions were freeze-dried, giving 830 mg of a freeze-driedproduct, which was purified by repeatedly subjecting it tohigh-performance liquid chromatography (column: μ Bondapak C₁₈, 40% v/vaqueous methanol 1 ml/min.) to give 32 mg of M-4 sodium salt and 280 mgof IsoM-4'sodium salt.

The properties of the IsoM-4'sodium salt were identical to those of theproduct of Example 12 and the properties of the M-4 sodium salt are asfollows:

(1) Nuclear Magnetic Resonance Spectrum:

Measurement was made at 200 MHz in deuteromethanol usingtetramethylsilane as the internal standard.

δ ppm:

0.91 (3H, triplet, J=7.5 Hz);

0.92 (3H, doublet J=7 Hz);

1.12 (3H, doublet, J=7 Hz);

1.1-1.8 (10H, multiplet);

2.25 (1H, doubled doublet, J=15 and 7.6 Hz);

2.34 (1H, doubled doublet, J=15 and 5.5 Hz);

2.2-2.4 (3H, multiplet);

2.48 (1H, multiplet);

3.68 (1H, multiplet);

4.07 (1H, multiplet);

4.28 (1H, multiplet);

5.36 (1H, multiplet);

5.48 (1H, doubled doublet, J=3 and 2 Hz);

5.88 (1H, doubled doublet, J=9.6 and 5.3 Hz);

5.98 (1H, doublet, J=9.8 Hz).

(2) Ultraviolet absorption spectrum (methanol solution) λ_(max) nm:

230.0; 237.2; 245.0.

(3) Infrared absorption spectrum (KBr) ν cm⁻¹ :

3400, 2900, 1725, 1580.

(4) Thin layer chromatography:

TLC plate: Merck silica gel Art 5715;

Solvent: benzene, acetone and acetic acid (50:50:3 by volume);

Rf value: 0.45.

EXAMPLE 15

18 mg of M-4 methyl ester were prepared, following the same proceduresas in Example 13, but using Cunninghamella echinulata IFO 4445.

EXAMPLE 16

33 mg of M-4 methyl ester were prepared, following the same procedure asin Example 13, but using Streptomyces roseochromogenus NRRL 1233.

EXAMPLE 17

12 mg of M-4 methyl ester were prepared, following the same proceduresas in Example 13, but using Syncephalastrum racemosum IFO 4814.

EXAMPLE 18

16 mg of M-4 methyl ester were prepared, following the same proceduresas in Example 13, but using Syncephalastrum racemosum IFO 4828.

EXAMPLE 19 Preparation of M-4 methyl ester

Five beagles (male, average weight 10 kg) were administered with ML-236Bat a dose of 200 mg/kg/day and their urine was collected for 3 days. 3liters of collected urine were passed through a 500 ml XAD-2 column,eluted with 500 ml of 50% v/v aqueous acetone, and, after distilling offthe acetone under reduced pressure, the residual liquid was adjusted topH 3 by the addition of trifluoroacetic acid. The mixture was thenextracted three times, each time with 1 liter of ethyl acetate, to giveM-4. This compound shows an Rf value of 0.45 on thin layerchromatography (TLC plate: Silica Gel Art 5715 manufactured by Merck &Co., Inc.: solvent: a 50:50:3 by volume mixture of benzene, acetone andacetic acid). The extract was washed with a saturated aqueous solutionof sodium chloride, and, after adding an ethereal solution ofdiazomethane, left standing for 30 minutes. It was then evaporated todryness under reduced pressure. The residue was dissolved in 10 ml of a55% v/v aqueous methanol solution, and passed through a columnchromatograph (Product of Merck & Co. Inc.; RP-8, Size B). After passing200 ml of a 55% v/v aqueous methanol solution, it was eluted with a 60%v/v aqueous methanol solution. The first 240 ml of the eluate werediscarded, and the next 120 ml were collected. This fraction wasevaporated to dryness and the residue was dissolved in 2.5 ml of a 65%v/v aqueous methanol solution and purified by high-performance liquidchromatography (JASCO-Trirotar, column: μ-Bondapak C₁₈). The portionwhich showed the fourth peak was separated and the solvent was distilledoff to give M-4 methyl ester as a colourless oil having the propertiesshown in Example 13. 4 mg of product were obtained.

EXAMPLE 20 Preparation of M-4

Homogenized rabbit liver was used in this Example to obtain M-4 fromML-236B.

(a) Enzymatic solution

Three volumes of a 1.15% w/v potassium chloride-10 mM phosphate (pH 7.4)buffer solution were added to one volume of rabbit liver and the mixturewas homogenized. The homogenized mixture was then centrifuged for 20minutes at 9,000 xg and the supernatant fraction was taken as anenzymatic solution. (b) Cofactor solution

    ______________________________________                                        B-nicotinamide adenine     3     mg                                           dinucleotide phosphate, reduced form (NADPH)                                  MgCl.sub.2 solution (508 mg/10 ml)                                                                       0.1   ml                                           1.15% w/v KCl solution     0.3   ml                                           0.2 M phosphate buffer solution (pH 7.4)                                                                 0.6   ml                                           ______________________________________                                    

The above substances were mixed to a total volume of 1 ml to make thecofactor solution.

(c) Reaction solution

80 μl of the above enzymatic solution, 20 μl of the above cofactorsolution and 2 μl of a methanol solution of ML-236B were mixed to make afinal concentration of ML-236B of 1 mM. The resulting solution wasshaken for 30 minutes at 37° C. M-4 was formed in the reaction mixtureand identified by TLC (the same conditions as in Example 19).

EXAMPLE 21 Preparation of M-4 sodium salt

2 mg of M-4 methyl ester were dissolved in 1 ml of a 0.1 N aqueoussolution of sodium chloride and subjected to hydrolyzation at 30° C. for1 hour. The reaction mixture was washed with 1 ml of chloroform and theresulting aqueous phase was adjusted to pH 8 with 0.1 N hydrochloricacid and passed through a XAD-2 column (about 5 ml). The column waswashed with 20 ml of distilled water and the desired product was elutedwith 15 ml of 50% v/v aqueous acetone. The acetone was distilled offfrom the eluate. The residue was confirmed by high-performance liquidchromatography to give a single peak (retention time was 13 minutes,eluted with 40% v/v aqueous methanol at 1 ml/minute). The residue wasthen lyophilized to give 0.8 mg of M-4 Na salt having the sameproperties as the product of Example 14.

EXAMPLE 22 Preparation of M-4 methyl ester

Each of twenty 500 ml. Evleumeyev flasks containing 100 ml. of a mediumhaving the composition listed below was inoculated with spores of Mucorhiemalis f. hiemalis IFO-5834. The inoculum was subjected to shakingculture at 26° C. and 220 rpm. After 4 days, ML-236B was added to afinal concentration of 0.05% w/v, and cultivation was conducted at 26°C. and 220 rpm for additional 6 days. The composition of the medium was(percentages are w/v):

    ______________________________________                                        Glucose        1.0%                                                           Peptone        0.2%                                                           Meat extract   0.1%                                                           Yeast extract  0.1%                                                           Corn steep liquor                                                                            0.3%                                                           Tap water      balance                                                                       (pH unadjusted).                                               ______________________________________                                    

After completion of the cultivation, the filtrate was adjusted to a pHof 3 with trifluoroacetic acid. The mixture was then extracted threetimes, each time with 100 ml. of ethyl acetate. There was obtained afraction containing M-4. M-4 has an Rf value of 0.45 on thin layerchromatography (Plate: Merck Silica gel Art 5715; Solvent: a 50:50:3 byvolume mixture of benzene, acetone and acetic acid). The conversionratio was 90%. This extract was washed with a saturated aqueous solutionof sodium chloride, after which there was added an ethereal solution ofdiazomethane. The resulting mixture was allowed to stand for 30 minutesand then concentrated under reduced pressure to dryness. The residue wasplaced on a Lobar column (Merck Si 60, size A) and purified with a 1:1by volume mixture of benzene and ethyl acetate. There were obtainedabout 600 mg. of M-4 methyl ester, having the same properties as theproduct of Example 13.

EXAMPLE 23 Preparation of M-4 lactone

The procedure described in Example 22 was repeated up to and includingwashing of the three ethyl acetate extracts with a saturated aqueoussolution of sodium chloride. The resulting solution was then evaporatedto dryness to give a lactone product. The product was recrystallizedfrom ethyl acetate to give about 560 mg. (56%) of M-4 lactone, havingthe same properties as the product of Example 1.

EXAMPLE 24 Preparation of M-4 sodium salt

The procedure described in Example 22 was repeated to give 1.9 liters ofthe filtrate from the conversion reaction. This was extracted threetimes, each time with 1 liter of ethyl acetate to give fractionscontaining M-4. By immediately transferring these into a 5% w/v aqueoussolution of sodium bicarbonate, there was obtained a fraction containingM-4 sodium salt. Then the M-4 sodium fraction was adjusted with 2 Nhydrochloric acid to a pH of 7.0 and adsorbed on an HP-20 column(manufactured by Mitsubishi Chemical Industries). Washing with water andelution with 50% v/v aqueous acetone gave a fraction containing M-4sodium salt, from which there were obtained 570 mg. (52%) of afreeze-dried product, having the properties described in Example 14.

EXAMPLE 25 Preparation of M-4

The procedure described in Example 22 was repeated, except that thefollowing microorganisms were employed and the conversion to M-4 was asshown by the associated codes:

    ______________________________________                                        Microorganism:       Conversion to M-4                                        ______________________________________                                        Mucor hiemalis f. hiemalis IFO-5303                                                                +4                                                       Mucor hiemalis f. hiemalis IFO-8567                                                                +4                                                       Mucor hiemalis f. hiemalis IFO-8449                                                                +4                                                       Mucor hiemalis f. hiemalis IFO-8448                                                                +4                                                       Mucor hiemalis f. hiemalis IFO-8565                                                                +4                                                       Mucor hiemalis f. hiemalis CBS-117.08                                                              +4                                                       Mucor hiemalis f. hiemalis CBS-109.19                                                              +4                                                       Mucor hiemalis f. hiemalis CBS-200.28                                                              +4                                                       Mucor hiemalis f. hiemalis CBS-242.35                                                              +4                                                       Mucor hiemalis f. hiemalis CBS-110.19                                                              +4                                                       Mucor hiemalis f. hiemalis CBS-201.65                                                              +4                                                       Mucor bacilliformis NRRL-2346                                                                      trace                                                    Mucor circinelloides f. circinelloides                                                             +1                                                       IFO-4554                                                                      Mucor circinelloides f. circinelloides                                                             +1                                                       IFO-5775                                                                      Mucor hiemalis f. corticolus NRRL-12473                                                            trace                                                    Mucor dimorphosporus IFO-4556                                                                      trace                                                    Mucor fragilis CBS-236.35                                                                          +1                                                       Mucor genevensis IFO-4585                                                                          +1                                                       Mucor globosus NRRL-12474                                                                          trace                                                    Mucor circinelloides f. griseo-cyanus                                                              +1                                                       IFO-4563                                                                      Mucor heterosoorus NRRL-3154                                                                       trace                                                    Mucor spinescens IAM-6071                                                                          trace                                                    Mucor chinensis IFO-4772                                                                           trace                                                    Rhizopus circinans ATCC-1225                                                                       + 1                                                      Rhizopus arrhizus ATCC-11145                                                                       +1                                                       Zygorynchus moelleri IFO-4833                                                                      +1                                                       Circinella muscae IFO-4457                                                                         +1                                                       Circinella rigida NRRL-2341                                                                        trace                                                    Circinella umbellata NRRL-1713                                                                     +1                                                       Circinella umbellata IFO-4452                                                                      +1                                                       Circinella umbellata IFO-5842                                                                      +1                                                       Actinomucor elegans ATCC-6476                                                                      +1                                                       Phycomyces blakesleeanus NRRL-12475                                                                trace                                                    Martierella isabellina IFO-6739                                                                    trace                                                    Gongronella butleri IFO-8080                                                                       +1                                                       Pycnoporus coccineus NRRL-12476                                                                    +3                                                       Rhizoctonia solani NRRL-12477                                                                      +2                                                       ______________________________________                                    

The codes representing the conversions to M-4 have the followingmeanings:

    ______________________________________                                        trace     =               0.5% or less                                        +1        =               0.5-5%                                              +2        =               5.0-10.0%                                           +3        =              10.0-30.0%                                           +4        =              70.0-90.0%.                                          ______________________________________                                    

EXAMPLE 26 Preparation of IsoM-4' lactone

Each of twenty 500 ml. Sakaguchi flasks containing 100 ml. of a mediumof the composition described in Example 22 was inoculated with spores ofCircinella muscae IFO-4457. The inoculum was subjected to shakingculture at 26° C. and 120 spm. After 4 days, ML-236B was added to afinal concentration of 0.05% w/v, and cultivation was conducted at 26°C. and 120 spm for a further 6 days.

After completion of the cultivation, the conversion reaction mixture wasfiltered, and the filtrate was adjusted with trifluoroacetic acid to apH of 3.0. The mixture was then extracted three times, each time with 1liter of ethyl acetate, giving a fraction containing IsoM-4'. Thisextract was washed with a saturated aqueous solution of sodium chlorideand then evaporated to dryness. There was obtained a lactone product.The residue was placed on a Lobar column (Merck: Si 60, size A) andpurified with an ethyl acetate system, giving 12 mg. of IsoM-4'lactone,having the properties described in Example 7.

EXAMPLE 27 Preparation of M-4'lactone

Each of twenty 500 ml. Erlenmeyer flasks containing 100 ml. of a mediumhaving the composition shown below was inoculated with spores ofSyncephalastrum nigrigans NRRL-12478. The inoculum was subjected toshaking culture at 26° C. and 220 rpm. After 3 days, ML-236B was addedto a final concentration of 0.05% w/v and cultivation was carried out at26° C. and 220 rpm.

The composition of the medium was as follows (percentages are w/v):

    ______________________________________                                        Glucose               1%                                                      Peptone             0.2%                                                      Meat extract        0.1%                                                      Yeast extract       0.1%                                                      Corn steep liquor   0.3%                                                                          (pH unadjusted).                                          ______________________________________                                    

After completion of the cultivation, the conversion reaction mixture wasfiltered, and the filtrate was adjusted with trifluoroacetic acid to apH of 3. The mixture was then extracted three times, each time with 1liter of ethyl acetate to give a fraction containing M-4', which has anRf value of 0.46 on thin layer chromatography (Plate: Merck silica gelArt 5715; solvent: a 50:50:3 by volume mixture of benzene, acetone andacetic acid). This extract was washed with a saturated aqueous solutionof sodium chloride, dried over anhydrous sodium sulphate and subjectedto lactonization by adding a catalytic amount of trifluoroacetic acid.The resulting mixture was then washed with a 5% w/v aqueous solution ofsodium bicarbonate, dehydrated with anhydrous sodium sulphate andevaporated to dryness. The residue was crystallized from ethyl acetateto give about 180 mg. of M-4'lactone having the following physicalproperties:

(1) Nuclear Magnetic Resonance Spectrum:

Measured in deuterochloroform at 100 MHz, using tetramethylsilane as theinternal standard.

δ ppm:

6.01 (1H, doublet);

5.90 (1H, quartet);

5.75 (1H, multiplet);

5.50 (1H, multiplet);

4.60 (1H, multiplet;

4.25 (1H, multiplet).

(2) Ultraviolet Absorption Spectrum (methanol)λ_(max) nm:

230, 237, 245.

(3) Infrared Absorption Spectrum (KBr)νcm⁻¹ :

3500, 1720.

(4) Mass spectrum:

M/e: 406(M⁺), 304, 286.

(5) Optical rotation:

[α]_(D) ²⁵ =+310.9° (c=0.66, methanol).

(6) Melting point:

141°-143° C.

(7) Elemental analysis:

Calculated: C, 67.95%; H, 8.43%

Found: C, 68.05%; H, 8.37%.

(8) Thin Layer Chromatography:

TLC plate: Merck silica gel Art 5715.

Solvent: Benzene-acetone (1:1 by volume)

Rf value 0.64.

EXAMPLE 28 Preparation of M-4' sodium salt

Following substantially the same cultivation procedures as in Example27, there was obtained a conversion reaction mixture.

After completion of the cultivation, the conversion reaction mixture wasfiltered, and the filtrate was adjusted with trifluoroacetic acid to apH of 3. It was then extracted three times, each time with 1 liter ofethyl acetate to give a fraction containing M-4', which was washed witha saturated aqueous solution of sodium chloride and immediatelythereafter passed into a 5% w/v aqueous solution of sodium bicarbonate,to give a fraction containing M-4' sodium salt. The aqueous layer thusobtained was adjusted to pH 8.0 with 0.1 N hydrochloric acid andadsorbed on a Diaion HP 20 resin column (manufactured by MitsubishiChemical Industries). It was then eluted with 50% v/v aqueous acetone.The acetone was distilled off, and the residue was freeze-dried to give1.41 g. of M-4' sodium salt, having the following physical properties:

(1) Nuclear Magnetic Resonance Spectrum:

Measured in deuterochloroform at 60 MHz, using tetramethylsilane as theinternal standard.

δ ppm:

600 (1H, doublet);

5.95 (1H, quartet);

5.70 (1H, broad singlet);

5.50 (1H, broad singlet).

(2) Ultraviolet Absorption Spectrum (methanol)λ_(max) nm:

230, 238, 246.

(3) Infrared Absorption Spectrum (KBr)νcm⁻¹ :

3400, 2900, 1680.

EXAMPLE 29 Preparation of M-4' methyl ester

Following substantially the same cultivation procedures as in Example27, there was obtained a conversion reaction mixture.

After completion of the cultivation, the conversion mixture was filteredand the filtrate was adjusted with trifluoroacetic acid to a pH of 3. Itwas then extracted three times, each time with 1 liter of ethyl acetate.The combined extracts were washed with a saturated aqueous solution ofsodium chloride and then an ethereal solution of diazomethane was addedthereto. The resulting mixture was allowed to stand for 30 minutes andthen concentrated under reduced pressure to dryness. The residue waspurified using a Lober column (Merck RP-8, size A) and a 1:1 by volumemixture of benzene and acetone as the developing solvent. There wereobtained 150 mg. of M-4' methyl ester as a colourless oily substance,having the following properties:

(1) Nuclear Magnetic Resonance Spectrum:

Measured in deuterochloroform at 60 MHz, using tetramethylsilane as theinternal standard.

δ ppm:

6.01 (1H, doublet);

5.90 (1H, quartet);

5.75 (1H, broad singlet);

5.50 (1H, broad singlet);

3.70 (3H, singlet).

(2) Ultraviolet Absorption Spectrum (methanol)λ_(max) nm:

230, 238, 246.

(3) Infrared Absorption Spectrum (liquid film)νcm⁻¹ :

3400, 1730.

(4) Mass analysis:

Measurement was made after silylation withN,O,-bis(trimethylsilyl)trifluoroacetamide using a mass spectrometer,type D-300 manufactured by Nippon Electronics.

M/e: 654 (M⁺)

We claim:
 1. The compound having the formula (V): ##STR19##
 2. Thecompound having the formula (VII): ##STR20##
 3. The compound having theformula (XIII): ##STR21##